Reprogramming Caspase‑7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition

The ability to routinely engineer
protease specificity can allow us to better understand and modulate
their biology for expanded therapeutic and industrial applications.
Here, we report a new approach based on a caged green fluorescent
protein (CA-GFP) reporter that allows for flow-cytometry-based selection
in bacteria or other cell types enabling selection of intracellular
protease specificity, regardless of the compositional complexity of
the protease. Here, we apply this approach to introduce the specificity
of caspase-6 into caspase-7, an intracellular cysteine protease important
in cellular remodeling and cell death. We found that substitution
of substrate-contacting residues from caspase-6 into caspase-7 was
ineffective, yielding an inactive enzyme, whereas saturation mutagenesis
at these positions and selection by directed evolution produced active
caspases. The process produced a number of nonobvious mutations that
enabled conversion of the caspase-7 specificity to match caspase-6.
The structures of the evolved-specificity caspase-7 (esCasp-7) revealed
alternate binding modes for the substrate, including reorganization
of an active site loop. Profiling the entire human proteome of esCasp-7
by N-terminomics demonstrated that the global specificity
toward natural protein substrates is remarkably similar to that of
caspase-6. Because the esCasp-7 maintained the core of caspase-7,
we were able to identify a caspase-6 substrate, lamin C, that we predict
relies on an exosite for substrate recognition. These reprogrammed
proteases may be the first tool built with the express intent of distinguishing
exosite dependent or independent substrates. This approach to specificity
reprogramming should also be generalizable across a wide range of
proteases.